Abstract– The identification of adenine by surface enhanced Raman scattering (SERS) on different mineral phases of a Martian meteorite Dar al Gani (DaG) 670 has been adopted as a test to verify the capability of this technique to detect trace amounts of organic or biological substances deposited over, or contained in, extraterrestrial materials. Raman spectra of different phases of meteorite (olivine, pyroxene, and ilmenite), representative of Martian basaltic rocks, have been measured by three laser sources with wavelengths at 785, 632.8, and 514.5 nm, coupled to a confocal micro-Raman apparatus. Adenine deposited on the Martian meteorite cannot be observed in the normal Raman spectra; when, instead, meteorite is treated with silver colloidal nanoparticles, the SERS bands of adenine are strongly enhanced, allowing an easy and simple identification of this nucleobase at subpicogram level.
Raman spectroscopy has also been proposed as a tool to detect organic materials of interest in astrobiology (El Amri et al. 2005; Busemann et al. 2007; Steele et al. 2007) and for the investigation of the origin of primitive life in extraterrestrial environments (Edwards et al. 2005; Marshall et al. 2006; Alajtal et al. 2010). Recently, Muniz-Miranda et al. (2010) and Caporali et al. (2011) have shown that Raman spectroscopy could be used to detect organic or biological traces, e.g., as a probe of extinct or extant life, only by adopting an approach based on the surface enhanced Raman scattering (SERS) effect. In fact, as reported by Kneipp et al. (2006) and Schlücker (2011), SERS can provide a huge enhancement of the Raman signal of organic or biological compounds mediated by the interaction with silver, gold, or copper nanostructured surfaces. This technique does not require a particular spectrometer, but only the addition of a metal hydrosol, usually silver, in order to get the SERS effect.
To assess the reliable use of SERS for the in situ search for life traces on Mars, the SERS investigation of nucleic acids sprayed on Martian rocks is a necessary preliminary step.
Adenine provides strong and characteristic SERS signals (Kneipp et al. 1998; El Amri et al. 2003; Bell and Sirimuthu 2006; Muniz-Miranda et al. 2010). The quest for adenine is therefore a reasonable goal, even for the automatic robotic instrumentations that will be available for the Martian explorations (Parnell et al. 2007). However, even if the suitability of SERS spectroscopy in the identification of nucleobases adsorbed on Martian meteorite has already been reported (El Amri et al. 2004), the effect of the mineral substrate nature as well as the possibility to use this technique as in situ tool for direct investigation on Mars’ surface still remains to be assessed. In the present paper, we report on the results of a detailed SERS analysis on the mineral phases of a Martian meteorite (DaG 670 belonging to the shergottite type) where adenine has been deposited.
Aiming to find the optimal experimental conditions for the detection of adenine in a genuine Martian rock, the SERS technique has been employed on the three major mineral phases constituting the meteorite: olivine, pyroxene, and ilmenite. Three different laser excitation wavelengths have been used: two in the red-light region (632.8 and 785 nm) and one in the green-light region (514.5 nm). Furthermore, beside the traditionally prepared silver colloidal suspension (Creighton et al. 1979), used to obtain the necessary SERS enhancement, another one has been prepared by addition of LiCl and tested on meteorite surface. Actually, the presence of chloride anions, other than providing further SERS activation, extends the stability of the colloidal suspension preventing precipitation even in extreme conditions such as those experienced during interplanetary travels.
A thick slice of the DaG 670 meteorite (about 15 × 10 × 2 mm), provided by the Museo di Scienze Planetarie in Prato (catalog number MSP 1385), was polished with a fine to ultra-fine grained diamond slurry (minimum grain size 0.25 μm), ultrasonically cleaned with water, rinsed twice with bidistilled water and air dried. Then, a drop (∼1 mm3) of dilute (∼10−2 moldm−3) adenine (99%+ Sigma-Aldrich) water solution was deposited on the sample surface. Once the solvent was evaporated, a drop of silver colloidal nanoparticles was added, air dried again and investigated by micro-Raman spectroscopy. For each of the three main mineral phases (olivine, pyroxene, and ilmenite) the Raman spectra were recorded on at least four randomly chosen grains. Since different crystallographic orientations and slight compositional variations can provide different Raman spectra, as well as more or less intense fluorescence phenomena, spectroscopic measurements on the same crystals were replicated, in order to collect sets of data not affected by the variability of the substrate.
The Ag nanoparticles (nps) in colloidal suspension were prepared according to the Creighton procedure (Creighton et al. 1979). The Ag nanocolloid have been divided in two portions with or without addition of LiCl in small concentration (∼10−3 M). The presence of chloride ions ensures a stronger Raman enhancement (Dong et al. 2011) and increases the stability of the colloidal solution up to several months (Muniz-Miranda et al. 2010).
To evaluate the performance of the Raman technique for the aimed purposes, two different spectrometers, a Renishaw RM2000 (at the Dipartimento di Chimica of the Università degli Studi di Firenze) and a Horiba Jobin-Yvon LabRAM-IR (at the Fondazione Prato Ricerche) both equipped with a 1800 g mm−1 single holographic grating, and three different laser wavelengths were used. The Renishaw RM2000 was alternatively coupled to a 785 nm stabilized diode laser source (red-light region) or to a 514.5 nm Ar-laser source (green-light region); the Horiba Jobin-Yvon was coupled to a He-Ne laser source emitting at 632.8 nm (red-light region). The laser beams, whose powers ranged between 3 and 8 mW depending upon the laser source, were focused on the sample using a 50× objective lens resulting in a laser spot footprint of about 3 μm2. The acquisition time was <30 s without observing changes in the spectra features of adenine.
In both cases the Raman light was filtered by a double holographic Notch filter system and collected by air-cooled CCD detectors in the wavelength region of 200–1200 cm−1 or 260–1200 cm−1 depending upon the type of filter used. All spectra were calibrated at 520 cm−1 using a silicon wafer.
Results and Discussion
Textural and Mineral Features of DaG 670
According to Folco and Franchi (2000), the DaG 670 Martian meteorite, classified as a Martian basalt (shergottite), has a porphyritic texture with mm-sized brown olivine (Fo58–80) crystals set in a fine-grained pyroxene groundmass. The groundmass is mainly constituted by pigeonite (En56–66Wo9–13) with subordinate enstatite (En73–82Wo2–3) and augite (En48–50Wo31–36). Among opaque phases ilmenite is dominant, whereas chromite, titanian chromite, and merrillite can be considered accessory phases. Figure 1 shows an optical microscopy image of the DaG 670 sample showing its mineral and textural features. The most abundant and representative mineral species (olivine, pyroxene, and among opaque phases, ilmenite) were investigated by micro-Raman spectroscopy for the adenine detection and the results obtained are hereafter comparatively discussed.
No Raman bands attributable to adenine have been observed in adenine-added samples that have not been covered by Ag colloidal particles. The collected spectra closely resemble the ones reported by Frosch et al. (2007), Mikouchi and Miyamoyo (2000) and Hochleitner et al. (2004) about Martian meteorites. Only the peaks attributable to vibrational modes of the substrate minerals such as olivine (820 and 850 cm−1), pyroxene (402, 674, and 1008 cm−1), and ilmenite (broad peak at about 660 cm−1) are clearly detectable in the Raman spectra collected on the adenine-added colloid-free DaG 670 sample (spectra #1 in Figs. 2A–C).
Raman (SERS) Spectra
The addition of Ag-nps dramatically changes the appearance of the Raman spectra. In those collected in the areas of the sample corresponding to olivine and pyroxene, the adenine Raman modes are highly enhanced and overtake the signal coming from the substrate (spectra #2 in Figs. 2A and 2B). This is remarkable indeed considering that adenine is present only as adsorbate thin layer, while a much larger material amount contributes to the Raman intensity of the mineralogic substrate. In particular the peak at 735 cm−1, assigned to the ring-breathing mode (Giese and McNaughton 2002; Sackmann and Materny 2006; Muniz-Miranda et al. 2010), displays the strongest enhancement. Since adenine were detected in all the points investigated it sounds reasonable to consider an almost uniform surface coverage by adenine. If so, by considering that the area wetted by the solution is ∼20 mm2 and the laser spot is ∼3 μm2, the amount of sample responsible for the SERS spectra is at level of 10−12 to 10−13 g.
The mineral substrate does not appear to significatively affect the vibration frequency of the ring-breathing mode at 735 cm−1, which may therefore be considered diagnostic and used for the adenine identification.
On the contrary a less favorable SERS signal enhancement was observed on ilmenite. The 735 cm−1 peak is still detectable but, with an intensity lower than observed in the corresponding spectra collected on silicates (curve #2 Fig. 2C). The reduced SERS effect can reasonably be accounted in two ways: (1) adenine might be less tightly adsorbed on ilmenite or (2) silver nanoparticles present minor affinity toward this type of substrate, which may result in locally depleted nanoparticles. The latter is the most likely explanation since the adenine deposition procedure (see the Experimental section) provides an almost homogeneously coated surface, independently from the nature of the substrate. On the other hand, the amount of Ag-nps proves to be directly connected with the intensity of the SERS-related peaks. Figure 3A shows a portion of the meteorite sample after the addition of Ag-nps. The substrate is constituted by an olivine grain and variable sized clusters of silver nanoparticles appeared irregularly scattered on its surface. Figure 3B shows the Raman spectra collected on five points of this olivine grain. Points 1–5 are characterized by the same substrate (olivine) but differ for the abundance of Ag-nps, whose amount increases from point 1 to point 5. The different amount of Ag-nps strongly affects the Raman spectra: in the points free of Ag-nps the spectra is characterized by the olivine-related peaks alone (spectrum #1 Fig. 3); increasing the amount of the nanoparticles the intensity of the broad band at 230–250 cm−1 assigned to the Ag-Cl and Ag-N stretching modes increases. In the same way the intensity of the adenine-related peaks increases, while the ones related to olivine decrease (curves #2 to #5 Fig. 3).
When the substrate is a polymineralic, such as shergottite in this study, the distribution of the silver nanoparticles is strongly inhomogeneous. In fact, as displayed in Fig. 4, on silicate phases the Ag-nps tend to constitute large fractal aggregates leaving almost totally uncovered the areas corresponding to the oxides (ilmenite). This observation does not prove the complete absence of silver nanoparticles in such zones, but provides a qualitative evidence of the lower affinity of silver nanoparticles toward oxides with respect to the silicates. This behavior can reasonably account for the reduced SERS effect evidenced in Fig. 3.
Chloride Effect on SERS Spectra
As described in the Experimental section, the presence of chloride ions adsorbed on the silver nanoparticles improves the stability of the colloidal suspension, making it suitable for interplanetary experiments. However, adsorbed chlorides ions could interfere in the Ag/adenine interaction leading to the decrease of the overall technique sensitivity. In order to test the changes in the SERS enhancement due to the addition of LiCl to the metal hydrosol, Raman spectra were collected on olivine pyroxene and ilmenite using chloride-activated Ag colloid. The obtained spectra (Fig. 5) were compared with the spectra achieved by employing traditionally prepared colloids (Fig. 2) without significant differences regarding the detection of adenine.
The Raman spectra collected on olivine and pyroxene (curves #2 in Figs. 5A and 5B) clearly exhibit, other than the peaks attributable to the substrates, the marker band of adenine at 735 cm−1. On ilmenite, as previously observed, the signal assigned to adenine is still detectable, albeit with much weaker intensity (curve #2 in Fig. 5C) compared with the spectra recorded on olivine and pyroxene.
These observations provide evidence that the presence of chloride anions does not impair the detection of this nucleobase by means of micro-Raman spectroscopy, allowing the use of such long-lasting silver colloidal suspension for SERS investigation.
Laser Wavelength Effect
It is well known that Raman spectra of organic molecules and minerals can be heavily affected by the excitation laser wavelength (Alajtal et al. 2010). Fluorescence phenomena are generally enhanced if short wavelengths are employed. Also the relative intensities of the Raman peaks of well-known substances can be remarkably modified leading to misinterpretation of spectra collected using different excitation sources. Therefore, spectroscopic analyses, with different excitation wavelengths, have been carried out on the same sample areas of the meteorite. The resulting Raman spectra have been compared to determine the more appropriate excitation wavelength for the identification of both the substrate mineral phase and the adenine. Figure 6 shows the results obtained using a 785 nm laser source (spectra A–C) and a 514.5 nm laser source (spectra D–F) both before (curves #1) and after (curve #2) addition of silver nanoparticles. The spectra here displayed present close analogies with those recorded using a 632.8 nm laser line. Before adding silver nanoparticles no bands attributable to adenine have been detected in any mineral substrate. Instead, the occurrence of the marker band of adenine (735 cm−1) clearly occurs when Ag nanoparticles are added. As observed before, on ilmenite the SERS effect results strongly reduced as compared to silicates, independently from the type of laser lines used in the Raman excitation, confirming the hypothesis that this phenomenon could be related with the shortage of silver nanoparticles. It is also worth pointing out that, as observed by Frosch et al. (2007) on silicate phases of other shergottites, the green-light laser emission causes a much higher fluorescence with respect to the red-light one. This results in a reduced signal-to-noise ratio that, in several cases, prevents the correct interpretation of the Raman signals (see for example the curve #1 of Fig. 6E). However, even if in these cases the identification of the substrate is difficult, after deposition of silver nanoparticles, there is a strong SERS effect to allow the unambiguous identification of adenine (curve #2 of Figs. 6D and 6E).
Conclusions and Perspectives
We obtained experimental evidence of the SERS capability to facilitate detection of traces of nucleobases on rock surfaces. A portion of a genuine Martian material (the shergottite Dag 670) was used as substrate for the SERS investigation and the role of the mineral phases constituting the sample was also evaluated. The Raman bands of adenine were enhanced by the SERS effect allowing a clear identification of this nucleobase, added in traces on the meteorite sample surface, especially on silicate (olivine and pyroxene) substrate. The estimated adenine amount responsible for the SERS spectrum was estimate about 10−12 to 10−13 g. Both red- and green-light laser excitations are effective for this identification. However, due to the limited fluorescence, the red-light emission should be preferred for such kind of investigations. At the same time the addition of LiCl to silver colloid does not affect the analytical performance of the SERS technique, suggesting the use of this stabilized long-lasting colloidal suspension.
The experimental procedure here proposed could be adopted for in situ search of life traces on extraterrestrial sites. In fact, our SERS measurements do not substantially differ from those proposed for Mars and asteroids exploration apart spraying the silver colloidal nanoparticles on rocks sample before performing spectroscopic investigation. Moreover, the chloride-stabilized silver colloid used in this study proved to be stable for several months (Muniz-Miranda et al. 2010) allowing it to reach the Martian surface without collapse.
Studies aiming to determine the applicability of SERS technique to detect other biomolecules on different extraterrestrial matrixes are currently under systematic evaluation.
Acknowledgements— The authors would like to thank Regione Toscana for financial support of the project LTSP through the fund POR FSE 2007-2013 (Obiettivo 2, Asse IV). This work has also been financed by MIUR-PRIN 2008 “Primitive Extraterrestrial Material as clues to the origin and evolution of the Early Solar System.”